Diabetes mellitus (DM) is a complex disease with a combination of genetic and environmental factors contributing to its onset, progression, and severity. A subset of DM patients is prone to developing ketoacidosis despite lacking the typical features of autoimmune type 1 diabetes. The phenotypes of these patients with ketosis-prone diabetes (KPD), strongly implicate novel mechanisms of pancreatic 2-cell dysfunction as etiologic factors. Recently, our group has identified four distinct phenotypes of KPD in more than 400 patients, followed in a longitudinal research clinic. The assignment is based on the A2 classification system that defines KPD patients with high accuracy and good prediction for long-term clinical outcome. The four KPD subgroups are A+2-, A-2-, A+2+, and A-2+, with a frequency distribution of 20%, 20%, 10%, and 50%, respectively. The role of sequence variation in genes underlying the phenotypic differences of the four groups is largely unknown and presumed to involve genetic factors in 2-cell development, function, expansion and regeneration, as well as those involved in 2-cell mitochondrial and lipid metabolism. Given the complexity of potential etiologic factors underlying the KPD syndromes, as many as 1,000 genes in each KPD patient may require resequencing to systematically pinpoint causative factors. Current Sanger methods for resequencing candidate genes at this scale are too expensive, labor-intensive, and time consuming. The goal of this R21 proposal is to evaluate the feasibility of our next-generation, cyclic reversible termination (CRT) sequencing approach by targeting 1,000 candidate genes on high-density oligonucleotide chips. The rationale for selecting the candidate genes is based on those known to be involved in 2-cell development and function, those associated with type 1 or type 2 DM, and those involved in ketogenesis. Recently, we have described a novel paradigm in reversible terminator (RT) chemistry, which has intrinsic advantages over conventional RT's with potential of exceeding current read lengths. We have developed and characterized four, fluorescently-labeled, 3'-unblocked RTs using synthetic templates fastened on standard microscope slides to demonstrate the feasibility of targeted resequencing. Here, we propose development of a 1,000 gene chip utilizing our CRT approach to resequencing genomic DNA from 50 KPD patients. These data will be validated by sampling ~10% of the discovered variants using traditional Sanger sequencing to assess accuracy of the CRT method. The outcome of this research will result in a comprehensive assessment of the CRT technology, while identifying putative gene sequence variations in KPD patients, all of which could lead to an expanded R01 study to evaluate a two-tiered approach of case- control and case-case comparisons (e.g., for the 2- group: A- versus A+, and conversely for the A- group, 2- versus 2+).